The ability to solve the three dimensional structure of a protein in a rapid and inexpensive manner is instrumental to the Structural Genomics mission. Protein structures add valuable information to the basic science and clinical knowledge base. On the clinical side, these structures can be instrumental for illuminating the function of important disease related proteins, for identifying inhibitors or enhancers of key signaling proteins that influence human health or for identification of drug targets. Currently, bacterial expression systems are most commonly enlisted for structure studies using nuclear magnetic resonance (NMR) and X-ray crystallography. We have recently discovered an ACA sequence-specific endoribonuclease in E. coli called MazF. Our preliminary studies demonstrate that cells possessing MazF activity facilitate the expression of high levels of protein derived from mRNAs engineered without ACA sequences. In contrast, because of the abundance of ACA sequences in host cell mRNAs, only trace amounts of background cellular protein synthesis occurs. The aims of this RO1 proposal are based on the hypothesis that application of the distinctive properties of MazF endoribonuclease to yeast cell expression systems will enhance the efficient expression and recovery of an array of correctly folded eukaryotic proteins. The unique properties of MazF expression will be exploited for development of a yeast single protein production (SPP) system. These attributes should facilitate NMR and X-ray structural studies without having to implement a protein purification step and will likely allow for direct visualization of protein structures in living cells using NMR. Aim 1 proposes experiments for the development of a MazF-based SPP system in yeast Saccharomyces cerevisiae. Aim 2 applies the technology developed in yeast S. cerevisiae to Kluyveromyce's lactis in an effort to amplify product yields. In Aim 3, the physiological consequences of MazF expression on yeast cells will be studied in order to optimize expression using the SPP system. Finally, in Aim 4 the SPP system will be applied to a subset of eukaryotic proteins that are unfolded when expressed bacteria. Proteins that display proper folding upon Heteronuclear Single Quantum Coherence analysis will then be subjected to NMR for structural determination.